Fine Needle Aspiration Biopsy Cytology of Lymph Nodes: A Diagnostic Approach Based on Pattern Recognition

Clinical Features

Lymph node hyperplasia refers to a variety of morphologic reaction patterns of benign nodal components, which may coexist with each other. Histologically, the most frequently recognized form is follicular hyperplasia, but others include interfollicular or paracortical hyperplasia and sinus histiocytosis. Their etiologies are numerous and include a variety of benign and, to a lesser extent, malignant conditions. Viral infections and less frequently bacterial infections are among the causes, which include, less commonly, drug reactions and autoimmune disturbances. In many patients, a specific etiology is not recognized and hence the term nonspecific or reactive hyperplasia is used.

Most frequently, hyperplasia affects one or two physically approximated lymph nodes. Statistically, cervical nodes are the most common superficial location, followed by inguinal or axillary lymph nodes, but any nodal group may be affected. With systemic diseases, reactive lymph nodes may present or evolve into diffuse lymphadenopathy.

Clinically, a thorough history often aids in determining the underlying problem, especially when combined with a physical examination. Palpation of hyperplasic nodes typically reveals enlarged, firm, single, nonmatted nodes that are usually not painful to the patient.

Histologically, follicular hyperplasia is characterized by an expanded cortex due to numerous lymphoid follicles, with well-developed germinal centers characteristically showing variation in both dimension and contour and sharply delineated by a collar of small lymphocytes, which are circumferentially organized. The germinal centers are formed by a mixture of small and large centrocytes and centroblasts with irregularly shaped nuclei and variably prominent nucleoli held together by follicular dendritic cells. They include tingible-body macrophages and lymphocytes. Mitotic figures are often numerous. A morphologic variant of follicular hyperplasia is termed progressively transformed germinal centers. Histologically, large germinal centers are infiltrated by small lymphocytes in variable numbers, creating an indistinct border. Progressively transformed germinal centers cannot be specifically recognized in FNAB smears and distinguished from follicular hyperplasia in general.

Interfollicular hyperplasia is characterized by a diffuse or nodular histologic appearance. This is due to a proliferation of lymphoid cells in the parenchyma between follicles. Although small lymphocytes are still present, the number of larger lymphoid cells including immunoblasts is noticeably increased. Plasma cells may also be evident in this paracortical zone hyperplasia.

Cytopathology

FNAB smears reflect the increased numbers of lymphocytes and larger lymphoid cells in hyperplasia by producing usually extremely cellular smears. The lack of intercellular cohesion that exists among lymphocytes leads to the dispersed nature of the lymphoid cells.

A critical cytomorphologic attribute is the identification of a heterogeneous population that consists of an admixture of small mature lymphocytes and larger lymphoid cells with all stages of maturation present (see Figs. 3-1A-C ), resulting in a spectrum of lymphoid cells from lymphocytes to the largest lymphoid cells including lymphoblasts. Importantly, the small lymphocytes numerically predominate. As stated earlier, the high N:C ratios of most of the constituent cells lead to a basophilic appearance. Tingible-body macrophages with phagocytized nuclear debris in the cytoplasm may be numerous. These appear as round solitary cells with variably sized and shaped punctate basophilic material in their cytoplasm, with a small reniform or round nucleus with pale chromatin.

The smears are also characterized by the presence of a variable numbers of germinal centers or “lymphohistiocytic aggregates” that are derived from the follicle germinal centers (see Fig. 3-1B ) and consist of a mix of small and large centrocytes and centroblasts, lymphocytes, and tingible-body macrophages held together by a meshwork of dendritic reticulum cell cytoplasm. These germinal center tissue fragments have irregular and frayed margins, but their staining intensity varies from deeply basophilic to pale, depending on the proportions of small lymphocytes, larger centrocytes, and centroblasts and reticulum cells.

Differential Diagnosis

Three conditions that may closely simulate or actually represent hyperplasia in part are metastatic tumors in which the neoplastic cells are sparse, Hodgkin lymphoma, and follicular lymphoma. In metastases with partial replacement of the node and low numbers of dispersed malignant cells, the benign hyperplastic lymphoid cell population can obscure the tumor cells. This is especially true in lobular carcinoma of the breast, where tumor cells have round, occasionally indented nuclei with fine pale chromatin and a moderate amount of pale cytoplasm, and in small cell carcinoma, where there is at least focal cell cohesion with molding of nuclei that are irregularly angulated and possess dense chromatin.

One clue to the diagnosis of Hodgkin lymphoma is the finding of eosinophils and plasma cells and sometimes prominent histiocytes mixed with a population of benign lymphocytes, possibly including germinal centers. This should prompt a diligent search for Reed-Sternberg (RS) cells or their variants, especially at the edge of the smear.

Follicular lymphoma has small, three-dimensional neoplastic follicles simulating germinal centers. The centrocytic and centroblastic lymphoma cells are morphologically different from small lymphocytes with their larger sizes and irregular nuclear outlines and make up both the neoplastic follicles and the diffusely spread lymphoma.

Several specific benign conditions need to be included in the differential diagnosis (DD) of follicular hyperplasia. One of these is Castleman disease, of which there are two types. In the hyaline vascular type, the germinal center structures are smaller than in regular hyperplasia and consist of large mononucleated or multinucleated follicular dendritic cells with round to oval nuclei, finely dispersed chromatin, and no nucleoli. Small vessels may occasional be seen penetrating into these altered centers. Plasma cells and plasmacytoid elements are numerous in the plasmacytic type. Castleman disease usually has a specific imaging and clinical presentation. In toxoplasmosis, there are syncytial microgranulomas in a background of follicular hyperplasia.

Ancillary Diagnostic Studies

In the majority of instances, when the classic features of follicular hyperplasia are present, the diagnosis can be made on smears alone, but immunophenotyping can be performed when there is an apparent increase in the proportion of larger lymphoid cells and/or a known history of lymphoma. Follicular hyperplasia shows a mix of B and T cells in relatively equal proportion with a prominent CD10- and CD20-positive germinal center B-cell component, while CD4 cells predominate among the T-cell component. Both kappa and lambda light chains are present (i.e., the cell types are polyclonal and a monoclonal B-cell population is not identified).

Clinical Features

Also known as “giant lymph node hyperplasia,” Castleman disease comprises two or more related conditions that have in common prominent lymphadenopathy. Occurring mostly in adults, Castleman disease has two distinct clinical presentations: (1) as a solitary mass lesion when patients are asymptomatic and a mediastinal mass is detected incidentally during radiologic workup of an unrelated condition and (2) as multicentric disease, which is more commonly associated with systemic manifestations including anemia, fever, and hypergammaglobulinemia. However, patients may also present with cervical or axillary lymphadenopathy. Surgical resection is usually curative in the solitary form, whereas administration of immunosuppressive agents is favored in the multicentric disease, which may persist for years. Patients with Castleman disease may have an increased propensity to develop malignant lymphomas and a variety of stromal neoplasms.

There are two major histologic forms of Castleman disease: the more common hyaline vascular type and the plasma cell type. Although there is overlap, most patients with the solitary form of Castleman disease have the hyaline vascular variant, whereas the plasma cell type corresponds to multicentric presentation. Abnormal-appearing germinal centers in the hyaline vascular type are characterized by a marked reduction in the number of lymphocytes and a prominence of follicular dendritic cells. The latter are associated with prominent proliferation of small blood vessels with distinctly hyalinized and thickened walls. The follicles appear enlarged due to a marked expansion of the mantle zone. Numerous concentric rings of small lymphocytes surround these retrogressive germinal centers, and classically, one or more well-developed venules penetrate through this thickened mantle zone and pierce the germinal center, forming so-called “lollipop” follicles. In the plasma cell type, the germinal centers may be replaced by an eosinophilic substance, which somewhat resembles amyloid. The nodes are enlarged secondary to a tremendous proliferation of plasma cells in the interfollicular zone, and the plasma cells may show nuclear atypia including multinucleated forms. In both types of Castleman disease, the plasma cells are polyclonal.

Cytopathology

It may be challenging, as discussed by Mallik and colleagues, to render a specific diagnosis of Castleman disease on FNAB smears ( Figs. 3-2A and B ). Samples from the hyaline vascular type are generally hypercellular and composed largely of dispersed lymphocytes with a predominance of small mature-appearing cells and some plasma cells. Tingible-body macrophages and typical germinal centers are not seen, although intact germinal centers with a prominent “onion skinning” formed by a corona of small lymphocytes penetrated by capillaries mimic “lollipop” follicles. These represent two low-magnification clues to this diagnosis.

Very characteristically, dendritic cell variants with one or more large round to oval nuclei and moderate amounts of pale cytoplasm and small or occasionally large nucleoli are seen singly and in small, loosely cohesive, syncytial aggregates. Typically, their nuclear outlines are corrugated or irregular, chromatin is fine to coarse, and longitudinal grooves may be seen (see Fig. 3-2B ). Finally, the smear background may contain fragments of capillaries, at times with hylanized walls (see Fig. 3-2A ).

FNAB smears of the plasma cell type are dominated by dispersed plasmacytic lymphoid cells and plasma cells, which may vary from small to large. Although some may appear completely normal, others may manifest nuclear atypia in the form of hyperchromasia, distinct nucleoli, and multinucleation.

Differential Diagnosis

The DD includes a reactive nonspecific follicular hyperplasia. A key difference is the finding in Castleman disease of small atrophic follicles with obvious follicular dendritic cells mixed with small numbers of lymphocytes. Infrequently, one may also observe small blood vessels protruding at the periphery. Although plasma cells may be seen in nonspecific hyperplasia, they are not present in the numbers associated with plasmacellular Castleman disease.

Ancillary Diagnostic Studies

The plasma cells are polyclonal in flow cytometry, while the dendritic cells are characteristically positive for CD21 and CD35 in cell blocks and core biopsies and negative for CD15, CD30, and the other B-cell and T-cell surface markers.

Clinical Features

Toxoplasma gondii, an obligatory intracellular organism, causes one of the most common human protozoan infections in patients of all ages, including a congenital form. A diverse clinical spectrum may occur secondary to this infection depending on several factors, especially the age at time of infection, the immune status of the patient, and the route of transmission of the infection. Whereas the central nervous system is the site of major clinical manifestations in patients who are immunosuppressed, especially those with the acquired immune deficiency syndrome (AIDS), most infected immunocompetent individuals are completely asymptomatic or present with an acute infection as lymphadenitis. Most characteristically this involves enlargement of nodes in the neck, especially the posterior cervical chain. One or more enlarged nodes, usually firm and nontender to palpation, are noted. A minority of patients will have generalized lymphadenopathy. A small proportion will also suffer associated symptoms such as fever and headache.

Although it is distinctly rare to identify specific organisms in the histology of nodal tissue, a constellation of microscopic features strongly suggests this infection, which can be confirmed on serology. In addition to well-developed hyperplasia of the lymphoid follicles, small syncytia of histiocytes form non-necrotizing microgranulomas that are intimately associated with the periphery of the follicles or involve any portion of the follicle including the germinal centers. The final characteristic histologic component is a marked hyperplasia of so-called monocytoid B cells that distend the sinuses.

Cytopathology

The smears from lymph nodes involved by toxoplasmosis are usually hypercellular with a heterogeneous lymphoid population in which small lymphocytes predominate. In addition, germinal center tissue fragments (“lymphohistiocytic aggregates”) may be well developed due to the florid follicular hyperplasia, and, most characteristically, a variable number of dispersed microgranuloma will be present.

The microgranulomas are flat, monolayered syncytia of epithelioid cells, which have abundant, homogeneous, nonvacuolated cytoplasm and multiple, ovoid, clearly benign nuclei. These oval nuclei have a delicate membrane and fine, even chromatin and inconspicuous nucleoli. They lack the centrally indented, elongated nuclei typical of larger granulomas. Orell and colleagues have described the monocytoid B cells as having abundant pale cytoplasm and solitary large ovoid nuclei with pale chromatin. As emphasized by Viguer and colleagues, inflammatory multinucleated giant cells, thick or three-dimensional granulomas, cytoplasmic vacuolization, and necrosis are not found. On rare occasions, tachyzoites of toxoplasma will fill and distend the cytoplasm of scattered macrophages or be seen in the background similar to platelets.

Differential Diagnosis

The finding of the characteristic microgranulomas separates this infection from nonspecific hyperplasia. In addition, if one is truly lucky, tachyzoites may be recognized.

Clinical Features

Classic heterophile-positive infectious mononucleosis is secondary to infection by Epstein-Barr virus (EBV). EBV infections occur worldwide and mostly in the first 3 decades of life. In infants and young children, the vast majority are completely asymptomatic. In adolescents and young adults, clinical manifestations of infectious mononucleosis are much more likely to occur and vary from patient to patient but typically include pharyngitis, a low-grade fever, atypical lymphocytosis in the peripheral blood, and lymphadenopathy. Typically, the latter involves the posterior cervical nodes but may occur more predominately elsewhere or even be generalized. The lymphadenopathy in mononucleosis may be painful and associated with rather firm nodes, with the pain due to rapid enlargement of the node with stretching of the capsule. Some patients will develop splenomegaly, and splenic rupture is a major, potentially life-threatening complication. Therapy is generally supportive, and the disease spontaneously resolves within weeks to a few months.

Histologically, the lymph node architecture is preserved overall, although follicles tend to be small and relatively sparse due to tremendous expansion of the paracortical tissue by proliferating neutrophils, macrophages, plasma cells, and lymphocytes in the interfollicular zones. Most importantly, immunoblasts are well developed and may be individually dispersed or congregated. They may even form sheets in the interfollicular region, occasionally associated with necrosis. Some of these cells may resemble RS cells. Venules with large endothelial cells may also be present in the paracortex. The lymphoid proliferation is due to infection of the B cells by EBV and the secondary or reactive growth of T cells, especially of the suppressor type.

Cytopathology

As might be expected, FNAB smears are extremely cellular and dominated by a polymorphous lymphoid population, which includes a large proportion of immunoblasts and plasmacytoid cells while small lymphocytes still predominate. In later stages of viral lymphadenopathy, apoptosis can be prominent and tingible-body macrophages become more numerous with decreased cellularity.

Immunoblasts are large and typically possess a single nucleus with a smooth nuclear outline, fine chromatin, and a huge, centrally located nucleolus. The abundant cytoplasm is characteristically basophilic. Compared with reactive nonspecific follicular hyperplasia, the number of large lymphoid cells is greatly increased.

Differential Diagnosis

The presence of a prominent population of large lymphoid cells raises the possibility of a large B-cell lymphoma, but a spectrum of lymphoid elements including small, intermediate, and large centrocytes and centroblasts, as well as plasma and plasmacytoid cells, is present in the immunoblastic proliferation pattern of viral infections and infectious mononucleosis, in particular. The viral lymphadenitis of cytomegalovirus and herpes simplex virus is indistinguishable unless diagnostic viral inclusions are seen. The antiepileptic agent phenytoin requires an accurate clinical history to differential diagnosis (DD) viral infections. The presence of immunoblasts is not expected in the plasmacytoid form of Castleman disease.

Although it is worrisome to identify large numbers of immunoblasts predominating one or more adjacent microscopic fields, it is important to remember that one should never render an interpretation of malignant lymphoma only on the basis of a portion of the sample; rather, the entire specimen needs to be scrutinized. The finding of Reed-Sternberg–like cells may raise the possibility of Hodgkin lymphoma, but in this disease a predominance of large lymphoid cells including immunoblasts and an absence of eosinophils is not expected. Immunoblasts lack the bilobed hyperchromatic nuclei and sky blue macronucleoli of typical Reed-Sternberg cells.

Ancillary Diagnostic Studies

In most instances, ancillary diagnostic procedures are unnecessary because the combination of the clinical presentation, appropriate serology results, and picture of immunoblastic interfollicular hyperplasia in the smears is sufficient for the correct diagnosis of infectious mononucleosis. The monospot test may be negative for a period after symptoms occur, and if the clinical presentation or serology or smear pattern is not typical, flow cytometry will show the polyclonal population with a mixture of B and T cells. With IHC for the latent membrane protein-1 of EBV, the B cells are positive. They also manifest EBV-related ribonucleic acid (RNA) when examined by in situ hybridization.

Clinical Features

Better known as Rosai-Dorfman disease, this is an idiopathic benign condition that classically presents clinically with painless and massive enlargement of bilateral or, on occasion, unilateral cervical lymph nodes. On physical examination, the enlarged nodes may be huge, firm, and matted together due to the prominent pericapsular fibrosis. Although the lymphadenopathy is usually asymptomatic, in some patients the nodes are so large that they may compress adjacent vital structures, requiring steroid therapy or even surgical resection. The lymphadenopathy may be accompanied by fever and anemia. In a significant minority of patients, the disease also involves extranodal sites, most commonly the orbit and other head and neck regions. In fact, some patients may present with or even have disease limited to extranodal organs. Overall, the prognosis is excellent with spontaneous remission occurring in most patients. The disease occurs mostly in the first 2 decades of life with a distinct predilection to involve African Americans. Although the etiology of the sinus histiocytosis with massive lymphadenopathy is unknown, it may be related to a defect in immunoregulation of histiocytes.

The massive enlargement is related to a uniform and prominent distension of the nodal sinuses with variable loss of the normal architecture in histology. The sinuses are packed with lymphocytes, plasma cells, and characteristically, histiocytes, which have small solitary benign nuclei and abundant pale cytoplasm. The cytoplasm typically contains numerous intact lymphocytes and occasional plasma cells and neutrophils, secondary to phagocytosis, termed emperipolesis. The lymphoid follicles are poorly developed, and large numbers of benign-appearing plasma cells are present in the interfollicular regions. A marked fibrosis of the nodal capsule with extension into the surrounding soft tissues is characteristic, although the fibrous connective tissue typically does not extend into the lymph node parenchyma.

Cytopathology

FNAB smears are moderately to highly cellular and include a polymorphic lymphoid cell population with a marked predominance of small lymphocytes. Larger lymphoid cells are evident but less numerous than expected in follicular hyperplasia while plasma cells may be numerous. The diagnostic feature is the presence of numerous individually scattered large histiocytes that contain recognizable small lymphocytes or, less commonly, plasma cells and neutrophils within their cytoplasm ( Figs. 3-4A and B ). Due to the large number of these huge histiocytes with their abundant pale cytoplasm in a heterogeneous background of mainly lymphocytes and plasma cells, the scanning lens view may have a mottled appearance.

Differential Diagnosis

This includes other causes of sinus histiocytosis including dermatopathic lymphadenopathy with its melanin-laden macrophages, nonspecific histiocytosis related to previous surgery, and leprosy with its mycobacteria containing macrophages or “leprae” cells.

Ancillary Diagnostic Studies

The diagnostic histiocytes are positive for S-100 protein and CD68, whereas they are nonreactive for CD1a, in cell block IHC. The lymphoid population is made up of polyclonal B-cells and larger numbers of T-cells, as shown by flow cytometry.

Clinical Features

Dermatopathic lymphadenitis represents the enlargement of one or more lymph nodes in the direct drainage pathway of skin lesions. The cutaneous disorders may be inflammatory or neoplastic and are typically excoriated. The nodes are usually only moderately enlarged at best, painless, nontender, and firm.

Histologically, the lymph node architecture is well preserved with the parenchyma showing a mixture of follicular hyperplasia and paracortical expansion. The latter characteristically appears pale due to the presence of numerous histiocytic cells with abundant poorly stained cytoplasm, mixed with macrophages, interdigitating dendritic cells, and Langerhans cells. The cytoplasm of the macrophages may contain phagocytosed melanin pigment. Eosinophils may or may not be present in large numbers.

Cytopathology

FNAB smears are usually quite cellular and include a polymorphic lymphoid cell population with a predominance of small mature lymphocytes and histiocytes, some containing melanin, along with tingible-body macrophages and some germinal center tissue fragments. The other characteristic attribute is the presence of interdigitating dendritic cells.

The interdigitating reticulum cells have abundant pale cytoplasm and solitary nuclei varying from oval to elongated with delicate membranes; pale, finely granular chromatin; indistinct nucleoli; and, characteristically, irregular nuclear contours with folds, convolutions, grooves, and pseudoinclusions. The presence of numerous blood vessels with adherent histiocytic cells has been described as a characteristic feature by Iyer and colleagues.

Differential Diagnosis

The diagnosis is usually straightforward if the pigmented histiocytes are appreciated in the smears. The melanin containing histiocytes should not be confused with metastatic melanoma, in which the nuclei are larger, have conspicuous nucleoli, and often have frequent pseudoinclusions.

Ancillary Diagnostic Studies

By IHC on cell block, the histiocytes are CD68 positive, and the most characteristic cells, namely the interdigitating dendritic cells, are positive for S-100 protein and negative for CD1a, while they may or may not express CD68. The lymphoid population is made up of polyclonal B cells and consists largely of T cells on flow cytometry.

Clinical Features

This group of related disorders affects younger individuals ranging from newborn infants to those in early adulthood. Although the unifocal form primarily involves bones, especially the skull, lymphadenopathy may be the sole feature of the disease. Lymphadenopathy, at times generalized, is also a component of the Lettere-Siwe variant. Clinically, the nodes are variably but not massively enlarged, painless and nontender, and soft to firm in consistency.

Histologically, the sinuses are markedly expanded by an infiltrate of neoplastic Langerhans cells, which range in size and number of the nuclei they possess, with smaller uninucleate and larger multinucleated cells. The constituent nuclei are round and situated within abundant eosinophilic cytoplasm, which varies in quality from homogeneous to foamy to somewhat distinctly vacuolated. The Langerhans cells are admixed with eosinophils, which may be relatively sparse or dominate numerically. In more advanced disease, the infiltrate extends into the paracortical zones.

Cytopathology

FNAB smears tend to be cellular and dispersed ( Fig. 3-5A ). Although follicular hyperplasia may be present in an involved node, its characteristic components, the germinal center tissue fragments, are not represented well in the smears. Rather, the specimen consists of a polymorphic lymphoid population with a marked dominance of small lymphocytes mixed with eosinophils and Langerhans cells.

Langerhans cells vary in their size and the number of nuclei present. The nuclei are characteristic; they appear elongated and have fine, pale chromatin; inconspicuous nucleoli; and thin membranes that are irregular (see Fig. 3-5B ), with the typical indentations, folds, and especially grooves (see Fig. 3-5C ). In some specimens, the eosinophils are so numerous that they may obscure the neoplastic Langerhans cells, rendering evaluation of the Langerhans nuclei difficult.

Differential Diagnosis

The major DD is granulomatous inflammation, but Langerhans cells do not typically aggregate to form granulomas and their nuclei have much higher length-to-width ratios compared with epithelioid cells and feature longitudinal grooves. Finally, eosinophils are not usually prominent in most granulomatous processes.

Ancillary Diagnostic Studies

Langerhans histiocytosis is not a lymphoid proliferation. Thus when evaluated by flow cytometry it is polyclonal, with an admixture of T and B cells. The Langerhans cells themselves are positive in IHC for S-100 protein and CD1a.